Our general plan is to isolate clones (both random and specific) of X linked DNA from human DNA recombinant libraries. These clones will be used to investigate the molecular biology of X chromosome inactivation and as probes for linkage studies of X linked inherited disease. The basis for selection of random X linked clones will be Southern blot hybridization to DNAs from cell lines varying in their human X chromosome composition (1X vs 4X human fibroblast DNA; mouse-human hybrid cells with the human X). X linked clones should be distringuised from clones with autosomal DNA by both quantitative (IX vs 4X human fibroblast DNA) and qualitative (mouse-human hybrid vs mouse DNA) differences in hybridization patterns. Clones will be taken from cDNA and genomic libraries and preselected for unique sequence DNA, where necessay, by colony hybridization with whole cell human DNA. In some cases erichment for X linked clones will be attempted by colony hybridization with DNA from mouse-human hybrid cells containing the human X chromosome. The basis for selection of specific X linked genes will involve three approaches: colony hybridization of specific synthetic DNA probes for hypoxanthine phosphoribosyl transferase (HPRT) to cDNA and genomic DNA libraries; construction of a human DNA cosmid library to be screened by DNA transformation for the HPRT gene; and screening by Southern blot hybridization of cDNA clones to DNA from interspecific DNA mediated transformants for the human HPRT gene (mouse recipient transformed by human DNA). This laboratory has been engaged in biological studies of mammalian X chromosome inactivation for some time and the project described above is an extension of our work to the molecular level.